C/EBPβ通过pim-1介导足细胞损伤的作用机制  

Mechanism of C/EBPβmediates podocyte injury through pim-1

作　　者：程维丽 陈晓攀 齐媛媛 文璐[1] 王晓阳[1] CHENG Weili;CHEN Xiaopan;QI Yuanyuan;WEN Lu;WANG Xiaoyang(Department of Nephrology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院肾脏内科,450052

出　　处：《天津医药》2022年第3期230-235,共6页Tianjin Medical Journal

基　　金：河南省科技攻关计划(202102310054)。

摘　　要：目的探讨CCAAT增强子结合蛋白β(C/EBPβ)/丝氨酸/苏氨酸激酶1(pim-1)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)轴介导小鼠肾脏足细胞损伤的作用机制。方法体外培养及转染足细胞后分为Control组、siRNA-NC组(用siRNA-NC转染足细胞)、siC/EBPβ组(用siC/EBPβ慢病毒转染足细胞)、Vector-NC组(空载体转染足细胞)和pim-1-OE组(pim-1过表达慢病毒转染足细胞)。脂多糖(LPS)与腺苷三磷酸(ATP)刺激足细胞后分为LPS+ATP组、LPS+ATP+siRNA-NC组、LPS+ATP+siC/EBPβ组、LPS+ATP+siC/EBPβ+Vector-NC组和LPS+ATP+siC/EBPβ+pim-1-OE组。实时荧光定量PCR(qPCR)检测C/EBPβ和pim-1 mRNA水平。Western blot检测C/EBPβ、pim1、NLRP3、p20半胱氨酸蛋白酶(Caspase)-1、Gasdermin D(GSDMD)、p17白细胞介素(IL)-1β蛋白水平。酶联免疫吸附试验(ELISA)检测炎性因子IL-1β和IL-6水平。染色质免疫共沉淀(chip)实验及双荧光素酶报告基因实验检测C/EBPβ与pim-1基因启动子结合。结果与Control组和siRNA-NC组相比,siC/EBPβ组的C/EBPβ及pim-1 mRNA水平明显下降(P<0.01)。与Control组相比,LPS+ATP组NLRP3、p20Caspase-1、p17IL-1β蛋白水平及IL-1β、IL-6水平明显升高(P<0.01)。与LPS+ATP+siRNA-NC组相比,LPS+ATP+siC/EBPβ组NLRP3、p20Caspase-1、p17IL-1β蛋白水平及IL-1β、IL-6水平降低(P<0.01)。chip实验及双荧光素酶报告基因实验显示C/EBPβ可与pim-1基因启动子结合。与Control组和Vector-NC组比较,pim-1-OE组pim-1 mRNA水平明显升高(P<0.05)。与LPS+ATP+siC/EBPβ+Vector-NC组相比,LPS+ATP+siC/EBPβ+pim-1-OE组NLRP3、p20Caspase-1、p17IL-1β、GSDMD蛋白水平及IL-1β、IL-6水平升高(P<0.05)。结论C/EBPβ/pim-1/NLRP3轴可能参与足细胞损伤,并作为LN治疗的潜在靶点。Objective To explore the mechanism of the CCAAT-enhancer-binding protein-beta(C/EBPβ)/serine/threonine kinase 1(pim-1)/NOD-like receptor thermoprotein domain-associated protein 3(NLRP3)axis mediating renal podocyte injury in mice.Methods After in vitro culturing and transfecting,mouse renal podocytes were divided into the Control group,the siRNA-NC group(podocytes were transfected with siRNA-NC),the siC/EBPβgroup(podocytes were transfected with C/EBPβlentivirus),the Vector-NC group(podocytes were transfected with empty vector)and the pim-1-OE group(podocytes were transfected with pim-1 overexpressed lentivirus).After podocytes were stimulated by lipopolysaccharide(LPS)and adenosine triphosphate(ATP),podocytes were divided into the LPS+ATP group,the LPS+ATP+siRNA-NC group,the LPS+ATP+siC/EBPβgroup,the LPS+ATP+siC/EBPβ+Vector-NC group and the LPS+ATP+siC/EBPβ+pim-1-OE group.C/EBPβand pim-1 mRNA were detected by real-time quantitative PCR(qPCR).The levels of C/EBPβ,pim-1,NLRP3,p20 cysteine protease(Caspase)-1,Gasdermin D(GSDMD)and p17 interleukin(IL)-1βwere detected by Western blot assay.The levels of IL-1βand IL-6 were detected by enzyme-linked immunosorbent assay(ELISA).Chromatin immunoprecipitation assay(chip)and dual luciferase reporter assay were used to detect the binding of C/EBPβto pim-1 gene promoter.Results Compared with the Control group and the siRNA-NC group,the level of C/EBPβand pim-1mRNA were significantly decreased in the siC/EBPβgroup(P<0.01).Compared with the Control group,the levels of NLRP3,p20Caspase-1,p17IL-1βprotein and IL-6 were significantly increased in the LPS+ATP group(P<0.01).Compared with the LPS+ATP+siRNA-NC group,the levels of NLRP3,p20Caspase-1,p17IL-1βprotein and IL-6 were significantly decreased in the LPS+ATP+siC/EBPβgroup(P<0.01).Chip assay and luciferase reporter assays showed that C/EBPβcould bind to pim-1 gene promoter.Compared with the Control group and the Vector-NC group,the level of pim1mRNA was significantly increased in the pim-1-OE group(P<0.05).Compar

关 键 词：狼疮肾炎 足细胞 NLR家族 热蛋白结构域包含蛋白3 CCAAT增强子结合蛋白β 原癌基因蛋白质cpim-1 

分 类 号：R593.242[医药卫生—内科学]